CN112903840B

CN112903840B - Application of serum metabolite in preparation of kit for diagnosing sjogren's syndrome

Info

Publication number: CN112903840B
Application number: CN202110065323.9A
Authority: CN
Inventors: 刘史佳; 陆燕; 郭云柯; 徐婷婷; 卢嘉微
Original assignee: Jiangsu Provincial Hospital of Chinese Medicine
Current assignee: Jiangsu Provincial Hospital of Chinese Medicine
Priority date: 2021-01-18
Filing date: 2021-01-18
Publication date: 2022-06-14
Anticipated expiration: 2041-01-18
Also published as: CN112903840A

Abstract

The invention discloses an application of a serum metabolite in preparing a kit for diagnosing Sjogren's syndrome. The serum metabolite beta-Mannosylglycete provided by the invention can be used for diagnosing and distinguishing healthy people and Sjogren syndrome patients, has high diagnosis accuracy, and has a prospect of being developed into a kit for diagnosing Sjogren syndrome.

Description

Application of serum metabolite in preparation of kit for diagnosing sjogren's syndrome

Technical Field

The invention belongs to the field of disease diagnosis, relates to discovery and application of disease diagnosis markers, and particularly relates to application of a serum metabolite in preparation of a kit for diagnosing sjogren's syndrome.

Background

Sjogren's Syndrome (SS) is an autoimmune disease characterized by invasion of salivary and lacrimal glands by lymphocytes, with B-cell hyperfunction. The main clinical manifestations of SS are dry eyes and mouth, and in addition to them, there are other symptoms of involvement of the exocrine glands and other organs outside the gland, which results in multiple system damage. SS includes both primary and secondary types, secondary to systemic autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus or systemic sclerosis. The prevalence rate of the primary SS is 0.3-0.7% in the population of China and 3-4% in the elderly. The disease is common in women, the incidence rate of men and women is 1: 9-20, and the incidence age is 40-50 years old.

At present, the pathogenesis of sjogren's syndrome is not clear, and no curative treatment is available. The clinical practice is mainly to take measures to improve symptoms, control and delay the progress of tissue and organ damage caused by immune response and secondary infection.

The biomarkers can be used as signal indicators reflecting the change of organism structure and function, and can be used for detecting the occurrence and the progression of complex diseases. In recent years, biomarkers in the field of omics are used as auxiliary means for judging the occurrence condition of diseases accurately and sensitively in advance, and a good effect is achieved. The combined diagnosis of multiple biomarkers can distinguish the type of the disease and the stage of the disease, and can assist clinical treatment. And taking a serum marker as an example, the method has the advantages of simplicity, rapidness, economy and relative non-invasiveness, is widely adopted and is very friendly to patients.

The invention finds a serum metabolite which can be used for diagnosing Sjogren's syndrome, and particularly provides the invention.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of the serum metabolite in preparing a kit for diagnosing Sjogren's syndrome.

The above purpose of the invention is realized by the following technical scheme:

use of a serum metabolite, wherein the serum metabolite is beta-mannosylglycine, for the preparation of a kit for the diagnosis of sjogren's syndrome, which is secondary sjogren's syndrome.

A kit for diagnosing Sjogren's syndrome, wherein the Sjogren's syndrome is secondary Sjogren's syndrome, and the kit contains a detection reagent for detecting serum metabolite beta-Mannosylglycerate.

Has the advantages that:

1. the diagnostic index provided by the invention is serum metabolite, can be detected only by adopting a small amount of blood, and is basically non-invasive;

2. the serum metabolite beta-Mannosylglycerate provided by the invention can effectively diagnose and distinguish healthy people and Sjogren syndrome patients, has high diagnosis accuracy, and thus has a prospect of being developed into a kit for diagnosing Sjogren syndrome.

Drawings

FIG. 1 is a graph comparing the levels of beta-Mannosylglycerate in the serum of healthy subjects and patients with Sjogren's syndrome;

FIG. 2 is a ROC curve for serum beta-Mannosylglycerate diagnosis differentiating dry syndrome vs in healthy subjects.

Detailed Description

The following detailed description of the present invention is provided in connection with the accompanying drawings and examples, but not intended to limit the scope of the invention.

Example 1: diagnostic efficacy of serum metabolites on sjogren's syndrome

First, experimental sample and reagent

225 healthy subjects and 210 patients with sjogren's syndrome were collected at the hospital of TCM in Jiangsu province. Healthy subjects were normal persons who were in physical health, and patients with sjogren's syndrome were diagnosed according to the classification criteria of American College of Rheumatology (ACR) sjogren's syndrome in 2012. The age, sex and body mass index of each group of patients were matched without significant difference. Each group of subjects or patients was randomly divided into training set samples and validation set samples as per table 1.

TABLE 1 training set sample and validation set sample numbers

	Healthy subjects	Patients with sjogren's syndrome
			Training set	135	125
Verification set	90	85
			Total number of samples	225	210

Exclusion criteria: firstly, the primary sicca syndrome patients and other autoimmune and chronic disease patients are diagnosed; ② serious primary diseases such as cardiovascular and cerebrovascular diseases, liver diseases, kidney diseases, hemopoietic system diseases and the like are combined; ③ the patients with mental diseases can not collaborate; pregnant or lactating women, or persons ready for pregnancy; adding other clinical testers within about 1 month; sixthly, the study is not accepted.

The main experimental reagents are as follows: liquid chromatographic grade Acetonitrile (ACN), methanol (MeOH), and methyl tert-butyl ether (MTBE) were obtained from merck, Germany (Darmstadt, Germany), and isopropanol, formic acid, and ammonium formate were obtained from ROE (DE, USA); the ultrapure water used was a Milli-Q system (Millipore, Billerica, MA, USA); n, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA), 1% Trimethylchlorosilane (TMCS), methoxyamine hydrochloride, pyridine and 1,2-¹³C₂Myristic acid was purchased from Sigma-Aldrich (st. louis, MO, USA).

Second, Experimental methods

1. Serum sample collection and storage

Collecting fasting peripheral blood of a patient in the early morning, placing the fasting peripheral blood in a test tube without anticoagulant, naturally agglutinating for 30-60min at room temperature, after blood coagulation, centrifuging at 2000rpm for 10min, carefully sucking supernatant clear serum liquid into a sterile freeze-drying tube, marking, and storing in a refrigerator at-80 ℃ for later use.

2. GC-MS technology for determining content level of each metabolite in serum

A detection instrument: performing chromatographic separation by using a Trace 1310 gas chromatograph together with an AS 1310 autosampler, performing full scanning on a TG-5MS gas chromatographic column by using a TSQ 8000 triple quadrupole mass spectrometer (Thermo Scientific, Waltham, MA, USA);

chromatographic conditions are as follows: the column was a TG-5MS gas chromatography column (0.25 mm. times.30 m, 0.25 μm). The carrier gas is He₂Setting a constant flow rate to be 1.2mL/min, adopting a flow dividing mode, setting a flow dividing flow rate to be 24.0mL/min, and setting a flow dividing ratio to be 20: 1, the temperature of a sample inlet is 250 ℃, and a temperature rise program is as follows: the initial temperature is 60 ℃, after the initial temperature is kept for 1min, the temperature is increased to 320 ℃ at the speed of 20 ℃/min, the initial temperature is kept for 5min, and the sample injection volume is 1 mu L.

The mass spectrometry method comprises the following steps: adopting EI mode, the temperature of ion transmission line is 250 deg.C, the temperature of ion source is 280 deg.C, the ionization energy is 70eV, the m/z collection range is 50-500, and the collection time is 3.5-19 min.

Sample treatment: the serum samples were thawed on ice, 225. mu.L of glacial methanol (containing 5. mu.g/mL of internal standard) was added to 40. mu.L of serum, after vortexing for 10s, 750. mu.L of glacial methyl tert-butyl ether (MTBE) was added, shaking was carried out at 4 ℃ for 10min, 188. mu.L of ultrapure water was added, after vortexing for 20s, centrifugation was carried out at 4 ℃ and 14000r/min for 2min, 350. mu.L of supernatant was aspirated into a 1.5mL centrifuge tube, evaporated to dryness, and stored at-20 ℃. The remaining liquid was centrifuged again for 2min, 250. mu.L of the lower layer liquid was aspirated, and 750. mu.L of methanol: 1-isopropyl alcohol: 1, vortex 15 s. Then, the mixture was shaken at 4 ℃ for 6min and centrifuged again for 2min at 14000 r/min. Finally, 475 μ L of the supernatant was taken in an EP tube, evaporated to dryness, one backup, one analysis. The dried lower layer material was added to 1. mu.L of 1,2-¹³C₂Carcoronic acid (internal standard, 5mg/mL) and 30. mu.L of a pyrimethanil solution (10mg/mL) were vortexed for 1min, shaken for 1.5h (30 ℃, 600r/min), and after adding 30. mu.L of BSTFA (containing 1% TMCS) again, vortexed for 1min, and shaken for 0.5h (37 ℃, 600 r/min). 50 μ L of the supernatant was taken and placed in a sample tube for GC-MS analysis. Sample analyses of SS and HC patients were run in random number order, with one QC inserted every 10 samples.

The content level is represented by the detection peak area of beta-mannosylglyceride in each sample.

3. Data processing method

In the training set, establishing a regression equation of the target serum metabolite by using Logistic regression to generate a group of new variables logit [ P ], carrying out ROC curve analysis on the new variables, and obtaining the optimal cut-off value according to the ROC curve; and in the verification set, the diagnosis accuracy of the target serum metabolite is calculated according to the prediction probability given by the SPSS25.0 software.

Third, experimental results

1. Differences in serum levels of target serum metabolites in healthy subjects and Sjogren's syndrome patients

In the training set, there was a significant difference in the levels of the metabolite beta-mannosylgyrate in the serum of healthy subjects and patients with sjogren's syndrome, as shown in figure 1.

2. Diagnostic discrimination potency of target serum metabolites on dry vs syndrome in healthy subjects

2.1 training set construction of binary logistic regression equation

In the training set, the content level of a serum metabolite beta-Mannosylglycerate of each sample is used as an independent variable, and a group (healthy subjects and Sjogren syndrome) is used as a dependent variable, so that a binary logistic regression equation logic [ p ] ═ 18.641+135.747X is constructed, wherein: x is the content level of beta-Mannosylglycerate.

2.2 training set determination of optimal discrimination thresholds

In the training set, the content level of the serum metabolite beta-Mannosylcerate of each sample is substituted into the binary logistic regression equation to obtain the regression value logit [ p ] of each sample in the training set, the possible regression value is used as a diagnosis point, the sensitivity and the specificity are calculated, and accordingly, an ROC curve (shown in figure 2) is drawn, the AUC is up to 1, and the accuracy is high. And calculating a Youden index according to the ROC curve, wherein the corresponding logit [ p ] value at the maximum value of the Youden index is 0.5 of the optimal cut-off value for diagnosing and distinguishing healthy subjects from Sjogren syndrome.

2.3 validation set validation diagnostic accuracy

In the verification set, the content level data of the serum metabolite beta-mannosylgyrate of each sample is introduced into SPSS25.0 software, so that a regression value logit [ p ] of each sample in the verification set can be obtained, a prediction probability is obtained, and the accuracy rate of the vs sicca syndrome of the target metabolite differentiated healthy subject is 100 percent (175/175) by dividing the number of correct samples by the total number of samples.

In conclusion, it can be seen that:

Example 2: diagnostic kit

The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.

Claims

1. Use of a serum metabolite, wherein the serum metabolite is beta-mannosylglycine, for the preparation of a kit for the diagnosis of sjogren's syndrome, which is secondary sjogren's syndrome.